Edited by Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved June 21, 2010 (received for review January 29, 2010) AbstractTelomeric repeat-containing RNA (referred to as TERRA), a noncoding RNA molecule, has recently been found in mammalian cells. The detailed structural features and function of the TERRA RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In the present studies, we investigated the structural features of human TERRA RNA in living cells. Using a light-switching pyrene probe, we found that human TERRA RNA forms a parallel G-quadruplex structure in living cells, providing the in vivo evidence for the presence of the G-quadruplex in human TERRA RNA. Furthermore, imaging experiments clearly show that TERRA RNA G-quadruplex localizes to telomere DNA at cell nuclei. These results provide valuable information to allow understanding of the structure and function of human TERRA RNA.telomere biologytelomere RNARNA foldingchemical probeTelomeres are essential structures at the ends of all eukaryotic chromosomes. In humans, telomeric DNA consists of a duplex region composed of TTAGGG repeats, ending in a shorter G-rich single-stranded overhang (1, 2). Telomeres play an important role in genome stability and cell growth by protecting chromosome ends (1, 2). Tumor formation and aging have been linked to alterations at the telomere (3, 4). Previous studies have suggested that human telomere DNA may exist in multiple states such as G-quadruplex or T-loop (5–7). For example, we along with two other groups determined the topology of human telomeric G-quadruplex in K+ solution (8–11). Our recent studies also demonstrated that such a (3 + 1) G-quadruplex can stabilize the T-loop structure (12).Telomeres have long been considered to be transcriptionally silent. A recent finding demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA in mammalian cells (13, 14). The telomeric repeat-containing RNA (referred to as TERRA) molecules were detected in different human and rodent cell lines, containing mainly UUAGGG repeats of heterogeneous length. TERRA RNA has not only been found in mammals but also in Saccharomyces cerevisiae (15). These findings raise the crucial question of how TERRA RNA is specifically associated with chromosome ends. The existence of TERRA RNA may reveal a new level of regulation and protection of chromosome ends that could facilitate valuable insight into fundamental biological processes such as cancer and aging. To reveal the structure and function of TERRA RNA will be essential for understanding telomere biology and telomere-related diseases.Recently, we and other two groups demonstrated that human TERRA RNA form G-quadruplex structures in the presence of Na+ and K+ solution by using NMR (16–18). However, whether TERRA RNA G-quadruplexes exist in living cells is unknown. We have developed photochemical approaches to probing different DNA structures for several years (19–22). For example, we detected diagonal loops in an antiparallel G-quadruplex based on the identification of photochemical products (20). Recently, using click chemistry, we successfully found that human telomere DNA and RNA sequences can form a DNA-RNA hybrid type G-quadruplex structure (23). Using a single-chain antibody, Schaffitzel et al. suggested that Stylonychia lemnae telomeric DNA forms an antiparallel G-quadruplex in vivo, in which about 2 × 108 telomeres are present in one macronucleus (24). Although these approaches gave some structural information, in humans the concentration of G-quadruplexes is too low to be detected in the presence of only a few dozen chromosome ends (92 telomeres/cell in G1 of the cell cycle and twice as many in G2). Nucleic acid structures are difficult to probe in vivo, and so far, direct evidence for human telomeric DNA and RNA G-quadruplexes existing in cells has not yet been obtained. Therefore, a more effective chemical method for probing G-quadruplex structure in living cells is desired.In the present studies, we designed and synthesized a light-switching pyrene probe to investigate whether TERRA RNA G-quadruplexes exist in living cells. A pyrene probe switching its fluorescence from monomer to excimer emission was used to detect G-quadruplex structure. Using this probe, we found that human TERRA RNA form a parallel G-quadruplex structure in living cells, providing in vivo evidence for the presence of G-quadruplexes in human TERRA RNA. Furthermore, colocalized-image experiments clearly showed that TERRA RNA G-quadruplexes localize at the cell nucleus. These results provide valuable information for understanding the structure and function of human TERRA RNA.Results and DiscussionDesign and Characterization of TERRA RNA G-Quadruplex-Specific Pyrene Excimer Probe.Using NMR spectroscopy, we have shown that TERRA RNA can form a parallel G-quadruplex structure in vitro (Fig. 1) (16), however, demonstration of their formation in living cells remains elusive. To investigate whether TERRA RNA G-quadruplexes exist in living cells, we employed a light-switching pyrene probe that has been designed to employ the capability of pyrene to form fluorescent excited-state dimers (excimers) (25–36). The advantage of the distance dependence of excimer formation with pyrene can be used as a unique excimer signaling device for detecting G-quadruplex structure (Fig. 2A). When the pyrene-labeled probe is free in solution without G-quadruplex formation, both pyrene molecules are spatially separated, and only the monomer emission peaks (at ∼400 nm) are observed. Formation of G-quadruplex brings the pyrene molecules at the 5′ and/or 3′ ends into close proximity, allowing the formation of an excimer. The excimer possesses broad red-shifted emission at ∼480 nm, in contrast with the pyrene monomer. The change in emission color serves as a way to rapidly probe G-quadruplex structure, and the excimer fluorescence intensity can be used for sensitive real-time monitoring of G-quadruplex formation.Download figureOpen in new tabDownload powerpointFig. 1. Schematic structure of human TERRA RNA G-quadruplex. A G-tetrad is formed by hydrogen bonds between adjacent guanines. Whether TERRA RNA G-quadruplexes exist in living cells is unknown.Download figureOpen in new tabDownload powerpointFig. 2. Design and characterization of TERRA RNA G-quadruplex-specific pyrene excimer probe. (A) Use of the pyrene excimer to probe G-quadruplex structure. The pyrene molecule has monomer emission near ∼400 nm. G-quadruplex formation brings the pyrene molecules close to each other. Consequently, pyrene excimer (green) forms, and green light (∼480 nm) is emitted after photoexcitation. (B) Fluorescence spectra for pyrene-modified oligonucleotide probes. (C) Chemical structures of pyrene probes with different linker lengths (L and R), RNA chain sequences, and numbers of pyrenes. E/M is the excimer/monomer fluorescence intensity ratio (E at 480, M at 380). (D) Response of excimer probe 5 to KCl solution. (E) Fluorescence image of probe 5 without (Left) and with (Right) KCl after illumination with a UV lamp (365 nm).We designed and synthesized a series of oligonucleotide probes having human TERRA RNA sequence and pyrene moieties at their 5′ and/or 3′ termini. To optimize excimer formation, the probes had varied linker lengths, RNA chain sequences, and numbers of pyrenes (Fig. 2C). We performed fluorescence microscopy experiments to investigate the excimer/monomer (E/M) fluorescence intensity ratio (Fig. 2B). Probe 5 bearing dual pyrene has the highest E/M ratio (E/M = 5.3) in the presence of K+ (Fig. 2C). The fluorescence spectrum of 5 in the absence K+ exhibits a monomer band at 400 nm, whereas the addition of K+ ions results in a strong excimer band at 480 nm, accompanying a decrease in monomer emission (Fig. 2D). The excimer assay was also carried out using buffer solution and cell nuclear extract. The probe 5 increased excimer fluorescence in nuclear extract compared to buffer solution (Fig. S1). This high sensitivity, together with fast measurements and detection, enabled visual detection of RNA G-quadruplex formation. A clear green color was observed by the naked eye when 200 mM of KCl was added to a 100 μL excimer probe solution (Fig. 2E). The CD spectrum of probe 5 in the presence of K+ at 25 °C showed a positive band at 265 nm and a negative band at 240 nm (Fig. S2), which are the characteristic CD signatures of a parallel G-quadruplex structure of RNA (37, 38). To further detect the topology of probe 5, we performed a CD spectroscopy experiment in nuclear extract (cell-like solution). The CD spectrum is similar to that in K+ solution (Fig. S2), suggesting a parallel topology with two linking loops positioned on the exterior of the G-quadruplex, consistent with the observations from CD experiments in K+ solution and NMR analysis (16, 17). Results from studies in simple solution and complex cell-extract systems using pyrene molecules are consistent with the structural model of TERRA RNA G-quadruplex demonstrated in the previous studies (16–18). These results suggest that the dual-pyrene probe could be used for probing TERRA RNA G-quadruplex formation in living cells.One distinct advantage of the light-switching excimer signaling method is that analytes can be detected without prior separation (25). Because only a G-quadruplex-formed probe gives excimer emission, the unformed probe does not have to be separated from the solution for G-quadruplex detection. Data shown in Fig. 2 C and D reveal that another advantage of this probe is that it enables ratiometric measurement. The G-quadruplex-formed probe gives three emission peaks, two monomer peaks at 375 and 398 nm, respectively, and an excimer peak at 480 nm. Signal fluctuation and impact of environmental quenching on the accuracy of the measurement could be effectively eliminated and minimized by taking the intensity ratio of the excimer peak to either one of the monomer peaks. This method can therefore be used for real-time detection and intracellular measurement.TERRA RNA G-Quadruplex Formation in Living Cells.To document direct evidence for the presence of TERRA RNA G-quadruplexes in living cell, dual-pyrene probe 5 was applied to living cells (Fig. 3A). We incubated human HeLa cells with 25 μM of probe 5 and visualized the live cells by fluorescence microscopy. We observed excimer fluorescence of pyrene using a 360 ± 40 nm excitation filter and a 470 ± 40 nm emission filter (green) (Fig. 3B). Negative cells that were not treated with probe 5 remained virtually nonfluorescent (Fig. 3B). To further verify G-quadruplex formation in living cell, two mutated probes 7 and 8 were used in the parallel experiments. In probe 7, all guanines were substituted with adenines. In probe 8, two guanine residues in different positions were substituted with adenines. Nonfluorescence was observed (Fig 3B and Fig. S3). These results demonstrate that the TERRA RNA G-quadruplex structure is present in vivo. Real-time response of the E/M ratio was investigated (Fig. 3C). The E/M ratio of probe 5 increased with time and then reached a plateau within 200 s, revealing that TERRA RNA G-quadruplex formation took place within seconds. Two control pyrene probes, 2 with only one pyrene at the 5′ end, and 7 with a random sequence, did not induce any excimer signal after the addition of KCl. We found that the E/M ratio of probe 5 reached a plateau within 600 s in nuclear extract and no-excimer signal in buffer control (Fig. S4), in comparison with KCl solution (within 200 s reached to plateau) (Fig. 3C), indicating a slow kinetics of G-quadruplex formation in nuclear extract. These results further suggest the usefulness of a light-switching excimer probe for real-time detection of RNA G-quadruplex structure in cellular environments. Next, we performed a time course of RNA G-quadruplex formation in live HeLa cells. After 0.5 h, we monitored the fluorescence response by microscopy (Fig. 3D). The excimer fluorescence increased with time; however, in contrast to the in vitro data, it slowly increased. These data indicate that (i) the transition of the pyrene probe occurs for some time in reaching a concentration sufficient for fluorescence visualizing, and (ii) like other RNAs, the probe is susceptible to enzymatic degradation. We find that TERRA RNA G-quadruplexes are stable in cells with a half-life  12 h (Fig. S5), consistent with previous observation that G-quadruplex formation induced an RNase resistance (16, 18). The pyrene molecules at the 5′ and 3′ termini of TERRA RNA may resist RNA degradation also.Download figureOpen in new tabDownload powerpointFig. 3. TERRA RNA G-quadruplex formation in living cells. (A) Schematic for detection of RNA G-quadruplex formation in living cells using dual-pyrene probe 5. G-quadruplex formation in living cells will induce the excimer fluorescence. (B) Fluorescence microscopy images of live cells. (iv–vi) HeLa cells were incubated with excimer probe 5, (vii–ix) probe 7, or (i–iii) buffer as a negative control. (C) Real-time response of excimer probe 5 and two pyrene-labeled control probes, 2 and 7, to 200 mM KCl. Probe 2 is a 5′-end pyrene-labeled TERRA RNA; 7 is a random sequence with pyrene labeled at both ends. (D) Time-lapse imaging of G-quadruplex formation in live cells using excimer probe 5.Some noncoding RNAs have been found to localize at chromatin as epigenetic gene regulators (39). To examine a possible relationship between TERRA RNA G-quadruplex and mammalian telomeric chromatin, we investigated the intracellular localization of TERRA RNA G-quadruplex using probe 5. The colocalized images between probe 5 and nuclear DNA clearly show that TERRA RNA G-quadruplex was restricted to the cell nuclei (Fig. 4A and Fig. S6). To further analyze the localization of TERRA RNA G-quadruplex, we performed telomere DNA fluorescence in situ hybridization (DNA FISH) experiment. Localization of telomeric DNA is shown using a fluorescently labeled CCCTAA repeat probe. The colocalization of RNA G-quadruplex (probe 5) foci and telomeric DNA foci was observed (Fig. 4B and Fig. S7). We then performed immunofluorescence experiment using antibodies against the human shelterin component TRF 2. TERRA RNA G-quadruplex foci colocalized with TRF 2 foci (Fig. 4C), indicating that TERRA RNA G-quadruplex is a common structural component of mammalian telomeres. We also detected TERRA RNA G-quadruplex at the ends of metaphase chromosomes from cells (Fig. 4D and Fig. S8), consistent with previous observations that accumulation of TERRA RNA is not only in nuclei but also at telomeres (13, 14). The association of TERRA RNA G-quadruplex with the ends of chromosomes suggests that telomere-related proteins may bind to TERRA RNA G-quadruplexes and promote the accumulation of TERRA RNA at telomere.Download figureOpen in new tabDownload powerpointFig. 4. Intracellular localization of TERRA RNA G-quadruplex. (A) Localization of TERRA RNA G-quadruplex at cell nuclei was determined by costaining using probe 5. Green signals correspond to the probe 5 (green). Cell nuclei were stained with SYTO 25 dye (red). The merged panel indicates a colocalization event. (B) Telomere DNA FISH experiments were performed to demonstrate that TERRA RNA localizes to telomeric DNA. TERRA RNA G-quadruplexes signals are in green (probe 5), telomeric DNA signals are red that are shown by situ hybridization using Cy5-labeled peptide nucleic acid probe, colocalization of the two signals generates orange signals in the merged panel. (C) Localization of TERRA RNA G-quadruplex was determined by costaining using antibodies anti human TRF 2. TERRA RNA G-quadruplexes signals are in green (probe 5), TRF 2 signals in red, colocalization of the two signals generates orange signals in the merged panel. (D) Detection of TERRA RNA G-quadruplex at the ends of metaphase chromosome. TERRA RNA G-quadruplexes signals are in green (probe 5); chromosome DNA was stained by propidium iodide (red).Possible Biological Significance.Clearly, we know a great deal about DNA G-quadruplex structures for G-rich sequences (40–43), but much less about RNA G-quadruplexes formed by more commonly existing single-stranded RNA. Recently, there have been several reports of the participation of RNA quadruplexes in gene regulation (37, 38). The finding of TERRA RNA molecules opens doors to better understanding of the essential biological role of telomeres. We have found that the TERRA RNA G-quadruplex in the presence of Na+ induced a strong RNase resistance for UUAGGG repeats in telomere RNA (16). There is a clear need to revisit the structural and functional mechanisms of telomeres accompanying TERRA RNA participation. We have focused considerable effort toward the identification of unique folding topologies for TERRA RNA architectures.TERRA RNA G-quadruplex has been found to localize to chromosome ends in cell nuclei, suggesting a possible association between TERRA RNA and telomere DNA. One possibility is that dimerization may occur on TERRA RNA G-quadruplex and DNA G-quadruplex. In fact, supporting this hypothesis, similar dimer structure formed by two telomere DNA G-quadruplex units has been suggested based on NMR and crystallography studies (6, 11). Some proteins have been suggested to be associated with TERRA RNA; a shelterin component, such as TRF2, was found to recruit TERRA RNA to telomeric DNA (44). We found that the nuclear extract can induce TERRA RNA G-quadruplex formation even in the presence of the metal inactivator EDTA, as well as this G-quadruplex structure associates with the ends of chromosomes, suggesting that the related proteins may exist in nuclei to bind TERRA RNA G-quadruplexes and accumulate TERRA RNA at telomere. Whether these proteins or other factors interact with the TERRA RNA G-quadruplex and facilitate it to chromosome ends remains to be discovered (45–47). The proposed structures might also have clinical relevance in the treatment of cancer, as the RNA molecule may contribute to the telomeric alterations accompanying malignant transformation (14). Thus, such G-quadruplex structures may be a valuable target for anticancer agents directed against telomeres (48–51). In this regard, it will be of great interest to evaluate the capacity of known DNA G-quadruplex ligands to bind to equivalent structures in TERRA RNA G-quadruplexes (52).The current study gives an insight into TERRA RNA structure in living cells. Undoubtedly, future studies should reveal potential connections between telomere protection, regulation, related proteins, and TERRA RNA. It remains to be established whether TERRA RNA G-quadruplex has a role in human telomere biology, or whether telomere-bound proteins have the ability to promote or disrupt RNA G-quadruplexes.MethodsCD Measurements.CD spectra were measured using a Jasco model J-725 CD spectrophotometer. The spectra were recorded using a 1-cm path-length cell. Samples were prepared by heating the oligonucleotides at 90 °C for 5 min and gradually cooling them to room temperature. In the CD melting studies, diluted samples were equilibrated at room temperature for several hours to obtain equilibrium spectra. Solutions for CD spectra were prepared as 0.3 mL samples at a 17 μM strand concentration in the presence of 100 mM KCl and 10 mM Tris·HCl (pH 7.0).Fluorescent Measurements.Fluorescent spectra were measured using a Jasco model FP-6500 spectrofluorometer. The spectra were recorded using a 1-cm path-length cell. For each sample, at least two spectrum scans were accumulated over a wavelength range from 360 to 600 nm. The excitation of the pyrene probe was achieved at 347 nm, the excitation wavelength of the pyrene monomer. The scan of the buffer alone was subtracted from the average scan for each sample. The measurement conditions were as follows: pyrene probe = 1 μM, Tris·HCl = 5 mM, KCl = 0 or 200 mM, pH 7.4, 20 °C. In photography experiments, UV irradiation of 365 nm was achieved with a UV Spot Light Source (Hamamatsu Photonics, 200 W) and UV-D36C filter (Asahi Technoglass) at 3.0 mW/cm2.Synthesis of Pyrene Probes.RNA oligoribonucleotide 5 was synthesized on 1-μmol scale 3′-Amino-Modifier C7 controlled pore glass (CPG) by conventional solid-phase synthesis (25, 30). After attaching 5′-Amino-Modifier C6 to the 5′ end, the resulting CPG was incubated with a mixture of pyrene acetic acid. After being stirred for 12 h at room temperature, the solution was removed and the resulting CPG was washed with dimethylformamide, water, and methanol (three times each). After incubation of the CPG with methylamine (50%) in ammonia at 65 °C for 10 min, the resulting supernatant was collected and evaporated to dryness. The dried pellet was diluted with water (1 mL), which gave a strong green fluorescence under UV irradiation. The solution was directly purified with RP-HPLC to give the desired product with two pyrene moieties at both the 5′ and 3′ ends, which was characterized with MALDI-TOFMS. [M - H+ calculated, 3951.9; found, 3950.6]. The probes (1–4, 6–8) were prepared using the above procedure.Cell Nuclear Extract Preparation.Nuclear extracts were prepared according to the published procedure (53). Briefly, 5 × 108 HeLa cells were suspended in 5 volumes of buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.1% Triton X-100, and a protease/phosphatase inhibitor mix; Roche). Nuclei were collected by centrifugation at 1,000  × g for 10 min. Isolated nuclei were extracted with 1 volume of buffer B (20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, and the protease/phosphatase inhibitor mix) on ice for 30 min. Extracts were further centrifuged at 11,000  × g for 30 min. The pellet was designated as the nuclear pellet; the supernatant was designated as the nuclear extract.Immunofluorescence.The cells that transfected dual-pyrene probe were washed in PBS, fixed for 15 min (room temperature) in 4% formaldehyde in PBS, and washed for 5 min with PBS three times. The cells were then dehydrated with cold methanol at RT for 10 min, washed, and blocked with blocking solution (3% skim milk, 0.05% Tween-20 in PBS) at RT for 1 h. The cells were blotted with 2 μg/mL antibody against mouse TRF 2 (Abcam) at 37 °C for 1 h. After washing three times, 5 μg/mL fluorescein (TRITC) goat anti-mouse (Abcam) as the secondary antibody in blocking solution was added and incubated at RT for 1 h. The fluorescence of antibody was observed using a fluorescent microscope with the excitation and absorbance filter of 546/10 and 600/40 nm.Cell Culture and Imaging Assay.HeLa cells (approximately 1 × 105) were seeded in a 35-mm dish (or approximately 2 × 104 on a glass slide) for 1 d for fluorescence microscopy experiments. Cultures were incubated at 37 °C and 5% CO2 in DMEM (1.5 mL) containing 10% FBS and antibiotics (penicillin and streptomycin). For transfection, the dual-pyrene probe (10 μL, 25 μM) in water without salt was diluted with DMEM (240 μL) without 10% FBS and antibiotics. LipofectAMINE 2000 reagent (Invitrogen) (10 μL) was activated in DMEM (240 μL) without 10% FBS and antibiotics by equilibration for 10 min at RT. The dual-pyrene probe and activated LipofectAMINE were mixed together, and the lipid complexes were incubated at 37 °C for 20 min. The lipid complexes were directly added to a 35-mm dish containing HeLa cells and mixed gently by rocking. The medium was removed, and the cells were then washed 5 times with PBS. For imaging the dual-pyrene probe, the excitation and absorbance filters were 360/40 and 470/40 nm, respectively, whereas they were 480/40 and 527/30 nm, respectively, for imaging nuclear DNA. Nuclear DNA staining was performed using SYTO 25 dye (molecular probe). The cells were incubated with SYTO 25 in DMEM (2.5 μM, 2 mL) without 10% FBS and antibiotics for 10 min at 37 °C in the presence of 5% CO2. For detection of telomeric DNA, the cells that transfected dual-pyrene probe were washed three times with HBSS and fixed with 4% PFA (paraformaldehyde) in PBS at RT for 10 min. Then cells were washed three times with PBS and dehydrated in 70%, 95%, and 100% ethanol for 5 min each. Cy5-(CCCTAA)3 peptide nucleic acid probe (100 nM) (Greiner Bio-One) in solution [10 mM Tris·HCl, 70% formamide, 0.5% blocking reagent (Roche), pH 7.2] was added on the cells, denatured at 80 °C for 3 min, and incubated at RT for 2 h in the dark. Then cells were washed washing solution 1 (10 mM Tris·HCl, 70% formamide, 0.1% BSA, pH 7.2) 2 times for 15 min each and washing solution 2 (100 mM Tris-HCl, 150 mM NaCl, 0.08% Tween-20, pH 7.2) 3 times for 5 min each. After dehydration in ethanol, the cells were observed with fluorescent microscope. To observation of metaphase chromosomes, the cells that transfected dual-pyrene probe were fixed to slides in 4% PFA in PBS or 3∶1 MeOH and glacial acetic acid (13). The slides were let dry in a fume hood after washing 3 times with PBS. DNA was stained with 1 μM propidium iodide. Adobe Photoshop software was used for image analysis.AcknowledgmentsThis work was partially supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports, Culture, and Technology, Japan (18001001) and by the Global Centers of Excellence Program for Chemistry Innovation.Footnotes1To whom correspondence may be addressed. E-mail: xuyan{at}mkomi.rcast.u-tokyo.ac.jp or komiyama{at}mkomi.rcast.u-tokyo.ac.jp.Author contributions: Y.X. designed research; Y.X., Y.S., and K.I. performed research; Y.X., Y.S., and M.K. analyzed data; and Y.X. and M.K. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1001177107/-/DCSupplemental. References↵Blackburm EH (2001) Switching and signaling at the telomere. Cell 106:661–673.OpenUrlCrossRefPubMed↵de Lange T (2004) T-loops and the origin of telomeres. 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We do not capture any email address.CAPTCHAThis question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Telomeric repeat-containing RNA structure in living cells Yan Xu, Yuta Suzuki, Kenichiro Ito, Makoto Komiyama Proceedings of the National Academy of Sciences Aug 2010, 107 (33) 14579-14584; DOI: 10.1073/pnas.1001177107 Telomeric repeat-containing RNA structure in living cells Yan Xu, Yuta Suzuki, Kenichiro Ito, Makoto Komiyama Proceedings of the National Academy of Sciences Aug 2010, 107 (33) 14579-14584; DOI: 10.1073/pnas.1001177107 Sign up for the PNAS Highlights newsletter to get in-depth stories of science sent to your inbox twice a month: Relatively clean snow and ice in the Indus River Basin during the COVID-19 pandemic may have reduced meltwater in 2020, compared with the 20-year average. Atmospheric and climate conditions could have created a cloud greenhouse effect to warm Mars and support liquid surface water. Researchers report a safety guideline to limit airborne transmission of COVID-19 that goes beyond the six-foot social distancing guideline. Interventions include using rice husks, manipulating paddy water and soil, and genetic changes that could stop arsenic from reaching the grain. Going beyond conventional approaches, researchers are using carefully cultured bacterial communities to improve sewage treatment.